Canadian Society of Clinical Chemists Harmonized Pediatric Lipid Reporting Recommendations for Clinical Laboratories.

Detecting dyslipidemia early is important because atherosclerosis originates in childhood and early treatment can improve outcomes. In 2022, the Canadian Cardiovascular Society (CCS)/Canadian Pediatric Cardiology Association (CPCA) published a clinical practice update to detect, evaluate, and manage pediatric dyslipidemia. However, guidance on its translation into clinical laboratories is lacking. The Canadian Society of Clinical Chemists Working Group on Reference Interval Harmonization Lipid Team aims to assist guideline implementation and promote harmonized pediatric lipid reporting across Canada. The 2022 CCS/CPCA clinical practice update, 2011 National Heart, Lung, and Blood Institute integrated guidelines, and new data analysis (Canadian pediatric reference values from the Canadian Laboratory Initiative on Pediatric Reference Intervals [CALIPER] and retrospective patient data from large community laboratories) were incorporated to develop 5 key recommendations. These include recommendations to: (1) offer nonfasting and fasting lipid testing; (2) offer a lipid panel including total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), non-HDL-C, and triglycerides, with apolipoprotein B and lipoprotein(a) available as individually orderable tests; (3) flag total cholesterol, LDL-C, and non-HDL-C results ≥ 95th percentile, and HDL-C results < 10th percentile, as recommended by CCS/CPCA/National Heart, Lung, and Blood Institute and validated by CALIPER, and flag apolipoprotein B and nonfasting triglyceride results ≥ 95th percentile on the basis of CALIPER, and do not flag lipoprotein(a) results but mention the adult cutoff in the interpretive comments; (4) implement interpretive comments listed in the current report; and (5) implement the National Institutes of Health LDL-C equation. The Canadian Society of Clinical Chemists Working Group on Reference Interval Harmonization Lipid Team will support clinical laboratories to implement these recommendations using knowledge translation strategies. Harmonizing pediatric lipid reporting across Canadian clinical laboratories will optimize clinical decision-making and improve cardiovascular risk management in youth.

The Present and Future of Lipid Testing in Cardiovascular Risk Assessment.

BACKGROUND: Lipids play a central role in the pathogenesis of cardiovascular disease (CVD), a leading cause of morbidity and mortality worldwide. Plasma lipids and lipoproteins are routinely measured to help identify individuals at high risk of developing CVD and to monitor patients' response to therapy. The landscape of lipid testing is rapidly changing, including new ways to estimate traditional lipid parameters (e.g., low-density lipoprotein-cholesterol [LDL-C] calculations) and new lipid parameters that show superiority for risk prediction (e.g., non-high-density lipoprotein-cholesterol [non-HDL-C], apolipoprotein B [apoB], and lipoprotein a [Lp(a)]). CONTENT: Various national guidelines for managing dyslipidemia to prevent CVD are available, which primarily focus on LDL-C for identifying those at high risk and setting thresholds for optimal response to therapy. However, LDL-C can be calculated and measured in various ways, each with advantages and disadvantages. Importantly, the recently established Sampson-NIH LDL-C equation appears to be superior to preceding calculations, as is clear from the literature and in guidelines. There is now a shift towards using lipid parameters other than LDL-C, such as non-HDL-C, apoB, and Lp(a), to identify high-risk patients and/or establish treatment targets. SUMMARY: The goal of this review is to discuss the present and future of lipid testing for CVD risk assessment through describing various national clinical guidelines, critically reviewing methods to calculate and measure LDL-C and discussing the clinical utility of additional lipid parameters.

Canadian Society of Clinical Chemists Harmonized Clinical Laboratory Lipid Reporting Recommendations on the Basis of the 2021 Canadian Cardiovascular Society Lipid Guidelines.

There is limited guidance on laboratory reporting and interpretation of lipids and lipoproteins used in cardiovascular risk stratification. This contributes to inconsistencies in lipid reporting across clinical laboratories. Recently, the Canadian Cardiovascular Society (CCS) published the 2021 CCS guidelines for the management of dyslipidemia for the prevention of cardiovascular disease in the adult. A subcommittee of the Working Group on Reference Interval Harmonization of the Canadian Society of Clinical Chemists has developed harmonized lipid reporting recommendations that are aligned with the 2021 CCS guidelines, to improve the standardization of lipid assessment and clinical decision-making. The proposed harmonized lipid reporting recommendations were critically reviewed by a broad range of laboratory and clinical experts across Canada. Feedback from approximately 30 expert reviewers was reviewed by the Working Group on Reference Interval Harmonization lipid subcommittee, and consensus decisions were incorporated into the 2021 harmonized lipid reporting recommendations. In this position statement, we provide 6 recommendations for laboratory reporting of lipid parameters. These recommendations include implementing the new National Institutes of Health equation to replace the Friedewald equation for calculating low-density lipoprotein cholesterol, offering lipoprotein (a), either as an in-house or send-out test, and using assays that report lipoprotein (a) in molar units (nmol/L). We also developed a harmonized lipid reporting format with interpretive comments that includes flagging results based on screening patients using treatment decision thresholds in a primary prevention setting. Overall, harmonized lipid reporting will help bridge the gap between clinical guideline recommendations and clinical laboratory reporting and interpretation, and will improve cardiovascular risk assessment across Canada.

Pediatric reference intervals for 1,25-dihydroxyvitamin D using the DiaSorin LIAISON XL assay in the healthy CALIPER cohort.

BACKGROUND: 1,25-dihydroxyvitamin D (1,25(OH)2D), the biologically active vitamin D metabolite, plays a critical role in calcium and phosphate homeostasis. 1,25(OH)2D is measured to assess calcium and phosphate metabolism, particularly during periods of profound growth and development. Despite its importance, no reliable pediatric reference interval exists, with those available developed using adult populations or out-dated methodologies. Using the fully automated chemiluminescence immunoassay by DiaSorin, we established 1,25(OH)2D pediatric reference intervals using healthy children and adolescents from the CALIPER cohort. METHODS: Serum samples from healthy subjects (0 to <19 years) were analyzed for 1,25(OH)2D using the DiaSorin LIAISON XL assay and age-specific reference intervals were established. The Mann-Whitney U-test was used to determine seasonal differences. Pooled neonatal and infantile samples were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine if elevated concentrations during the first year of life may be attributed to cross-reacting moieties. RESULTS: Three reference interval age partitions were required with highest levels in subjects 0 to <1 year (77-471 pmol/L), which declined and narrowed after 1 year (113-363 pmol/L) and plateaued at 3 years (108-246 pmol/L). 1,25(OH)2D concentration was not significantly affected by seasonal variation or sex. Elevated 1,25(OH)2D concentrations in neonatal and infantile samples may be the result of an interfering substance. The absence of 3-epi-1,25-dihydroxyvitamin D in the pooled samples makes it unlikely to be the interfering moiety. CONCLUSIONS: Pediatric reference intervals for 1,25(OH)2D were established to improve test result interpretation in children and adolescents. 1,25(OH)2D is elevated in a proportion of neonates and infants, which may be the result of a cross-reacting moiety.

Evaluation of the N-latex serum free light chain assay on the Siemens BNII analyzer and agreement with The Binding Site FreeLite assay on the SPAPlus.

OBJECTIVES: To evaluate the Siemens N-latex kappa free light chain (κFLC) and lambda FLC (λFLC) assays on the BNII nephelometer and assess agreement with The Binding Site Freelite FLC assays on the SPAPlus. DESIGN AND METHODS: Over 180 patient serum samples from routine analysis of κFLC and λFLC measured by the Freelite assay were collected for the study and measured with the N-latex κFLC and λFLC assays to assess precision, linearity, method comparison and dilutional effects. RESULTS: Complex precision showed coefficients of variation of 4.8-7.2% for the κFLC assay and 3.6-6.0% for the λFLC assay. Linearity assessment showed both assays were linear (κFLC, y=1.00x-0.09 and λFLC, y=1.050x-1.252). Qualitative method comparison showed 87.9% (116/132) agreement and Cohen's kappa of 80.4% between the κFLC assays and 72.6% (98/135) agreement and Cohen's kappa of 55.4% for the λFLC assays. Quantitative method comparison for κFLC<150mg/L was y=0.92x+2.21, R=0.661 and for λFLC<150mg/L was y=7.90x-137.96, R=0.526. Dilutional effects including antigen excess and non-linearity were also examined. CONCLUSIONS: The N-latex assay showed good precision and linearity with reasonable agreement to the Freelite assay. However, the assays should not be used interchangeably to monitor patients.

Pediatric Reference Intervals for Biochemical Markers: Gaps and Challenges, Recent National Initiatives and Future Perspectives.

Reference intervals provide valuable information to medical practitioners in their interpretation of quantitative laboratory test results, and are critical in the assessment of patient health and in clinical decision-making. The reference interval serves as a health-associated benchmark with which to compare an individual test result. While the concept of reference intervals and their utility appear straightforward, the process of establishing accurate and reliable reference intervals is considerably complex and involved. Currently, many pediatric laboratory tests are inappropriately interpreted using reference intervals derived from either adult populations, hospitalized pediatric populations, or from outdated and/or inaccurate technology. Thus, many pediatric reference intervals used in diagnostic laboratories are incomplete and may be inappropriate for clinical use. The use of inappropriate reference intervals impacts clinical decision-making and has potential detrimental effects on the quality of patient healthcare including misdiagnosis, delayed diagnosis, inappropriate treatments, and patient risk. These are critical gaps in pediatric healthcare and it is imperative to update and establish appropriate reference intervals for pediatric populations based on specific age- and sex-stratifications. In the present review, specific issues, challenges and deficiencies in pediatric reference intervals for biochemical markers will be discussed. Early studies using hospitalized patients will be examined, followed by a review of recent national and global initiatives on establishing reference intervals from healthy pediatric population. We will highlight the achievements and milestones of the Canadian CALIPER project, including the establishment of a comprehensive biobank and database which has addressed several of these critical gaps. CALIPER's mandate is to establish and provide comprehensive, up-to-date pediatric reference intervals to all biochemical markers of pediatric disease. CALIPER has also begun knowledge translation initiatives to disseminate its data via peer-reviewed publication, an online database, and a smartphone application to allow greater access to CALIPER pediatric reference interval data. Finally, limitations, future perspectives and harmonization of pediatric reference intervals to improve pediatric diagnostics in Canada and worldwide will be discussed.

Metformin Elicits Antitumor Effects and Downregulates the Histone Methyltransferase Multiple Myeloma SET Domain (MMSET) in Prostate Cancer Cells.

BACKGROUND: This study explored the biological effects of metformin on prostate cancer (PCa) cells and determined molecular pathways and epigenetic regulators implicated in its mechanism of action. METHODS: We performed mRNA expression profiling in 22Rv1 cells following 2.5 mM and 5 mM metformin treatment. Genes significantly modified by metformin treatment were ranked based on altered expression, involvement with cancer-related processes, and reported dysregulation in PCa. The effects of the top ranked gene, MMSET, on the proliferative and invasive capabilities of PCa cells were investigated via siRNA knockdown alone and also combined with metformin treatment. RESULTS: Metformin treatment decreased cell growth of PCa cell line 22Rv1 and stalled cells at the G1/S checkpoint in a time- and dose-dependent manner, resulting in increased cells in G1 (P < 0.05) and decreased cells in S (P < 0.05) phase. Metformin activated the AMPK/mTOR signaling pathway as shown by increased p-AMPK and decreased p-p70S6K. mRNA expression profiling following metformin treatment identified significant changes in 136 chromatin-modifying genes. The top ranked gene, multiple myeloma SET domain (MMSET) showed increased expression in PCa cell lines (22Rv1 and DU145) when compared to the benign prostate epithelium-derived cell-line RWPE-1, and its expression was decreased upon metformin treatment. siRNA-mediated knockdown of MMSET showed decreased cellular migration and invasion in DU-145 cells. MMSET knockdown in combination with metformin treatment resulted in further reduction in the capacity of PCa cells to migrate and invade. CONCLUSIONS: These data suggest MMSET may play a role in the inhibitory effect of metformin on PCa and could serve as a potential novel therapeutic target for PCa. Prostate 76:1507-1518, 2016. © 2016 Wiley Periodicals, Inc.

Integrated analysis of epigenomic and genomic changes by DNA methylation dependent mechanisms provides potential novel biomarkers for prostate cancer.

Epigenetic silencing mediated by CpG methylation is a common feature of many cancers. Characterizing aberrant DNA methylation changes associated with tumor progression may identify potential prognostic markers for prostate cancer (PCa). We treated two PCa cell lines, 22Rv1 and DU-145 with the demethylating agent 5-Aza 2'-deoxycitidine (DAC) and global methylation status was analyzed by performing methylation-sensitive restriction enzyme based differential methylation hybridization strategy followed by genome-wide CpG methylation array profiling. In addition, we examined gene expression changes using a custom microarray. Gene Set Enrichment Analysis (GSEA) identified the most significantly dysregulated pathways. In addition, we assessed methylation status of candidate genes that showed reduced CpG methylation and increased gene expression after DAC treatment, in Gleason score (GS) 8 vs. GS6 patients using three independent cohorts of patients; the publically available The Cancer Genome Atlas (TCGA) dataset, and two separate patient cohorts. Our analysis, by integrating methylation and gene expression in PCa cell lines, combined with patient tumor data, identified novel potential biomarkers for PCa patients. These markers may help elucidate the pathogenesis of PCa and represent potential prognostic markers for PCa patients.